Abstract Healthy human cardiac muscle is composed of two myosin isoforms, alpha and beta, which are significantly different in kinetics of the cross-bridge cycle. The specific myosin isoform composition tunes muscle for maximum performance, force production, power output, and economy. The mechanisms behind this fine muscle's performance tune-up are not known, thus limiting our understanding of a fundamental muscle property. Our long-term goal is to apply insights gained from learning the origin of kinetic differences of cardiac myosin isoforms to help devise ways to restore impaired muscle function. We envision tune-up of failed heart muscle by mimicking the healthy muscle isoform composition. The immediate objective of this proposal is to elucidate the molecular mechanism of kinetic difference of cardiac myosin isoforms. Our central hypothesis is that isoform kinetics i determined by the electrostatic interactions at the regulatory site within myosin head. Our hypothesis is based on a significant body of experimental data, confirming possibility of myosin kinetics modulation. Three specific aims are to validate our hypothesis. The specific aims are focused on potential regulatory sites within different parts of myosin head, Loop1 (Aim 1), upper 50 kDa domain (Aim 2), and the force generating region (Aim 3). According to our working hypothesis, these regulatory sites modulate myosin-nucleotide interaction, including the rate of ADP release from the cross-bridge, which is the rate limiting step of the slow beta myosin isoform. We plan to use site specific mutagenesis, altering electrostatic interactions within proposed regulatory sites. We will prepare mutants of recombinant human cardiac beta myosin and will characterize kinetics of prepared myosin mutants. We will examine our hypothesis on importance of electrostatic interactions within proposed regulatory sites on myosin kinetics modulation. The proposed research is significant because it will provide a detailed understanding of the mechanism by which human cardiac muscle is fine tuned for maximum performance. We will gain new insights into fundamental mechanisms by which produced force and power, and muscle energetics are modulated by muscle isoform composition. Elucidating kinetic tune-up mechanisms may lead to therapeutic ways of restoring or improving muscle function.